Proteases have been used in the industry for a long time. They are used in a variety of fields including detergents such as laundry detergents, fabric modifiers, leather processing agents, cosmetics, bath agents, food modifiers or medicines. Of these uses, proteases for detergents are industrially produced in the largest amount. Examples of proteases for detergents include Alcalase and Savinase (registered trademarks; Novozymes A/S), Maxacal (registered trademark; Genencor International Inc.), BLAP (registered trademark; Henkel KGaA), and KAP (Kao Corporation).
The purpose of incorporating protease into a detergent is to promote degradation of protein-based stains attached to clothing into low molecular Weight substances, and solubilization thereof by a surfactant. However, actual stains are complex stains which contain not only proteins but also a mixture of plural components such as organic substances and inorganic substances, for example, sebum-derived lipids and solid particles. Therefore, there is a demand for detergents having a significant detergency on such complex stains.
Based on this standpoint, the present inventors have found a few kinds of alkaline proteases, which retain a sufficient caseinolytic activity even in the presence of a high concentration of a fatty acid, exhibit a significant detergency on complex stains containing not only proteins but also sebum and the like weaved therewith, and have a molecular weight of approximately 43,000 (Patent Document 1). It is suggested that the aforementioned group of alkaline proteases are categorized to a novel subtilisin subfamily, unlike the conventionally known serine proteases typified by subtilisin derived from bacteria belonging to Bacillus sp., in terms of their molecular weights, primary structures, enzymatic properties, and, in particular, exhibiting extremely strong oxidant resistance (Nonpatent Literature 1).
The above group of alkaline proteases has a significant detergency under the conditions where sebum stains and the like are weaved with each other. Moreover, alkaline proteases having sufficient productive performances in addition to detergency remain in demand.
In general, an enzymatic activity changes in a pH-dependent manner because the enzyme has an amino acid residue having either a dissociable acidic group or a dissociable basic group, which is essential for the expression of the activity thereof, at the active site of the enzyme. Therefore, an attempt to artificially alter the pH-dependence of an enzymatic activity by altering a charged amino acid residue is one of the important targets in protein engineering.
For example, it is reported that substitution of a specific amino acid residue in subtilisin BPN′ leads to alteration in the pH-dependence by altering the surface charge of the enzyme (Non-patent Documents 2 to 4), M-protease which is a high-alkali resistant protease adapts to high-alkali conditions when the surface amino acid residues are altered to acquire a high isoelectric point and gain a new network of ion bonds (Non-patent Document 5), and that there is a constant relationship between the isoelectric point and the detergency of subtilisin (Patent Document 2).
However, any of the above mentioned reports relates to serine proteases derived from bacteria belonging to Bacillus sp., having a molecular weight of approximately 28,000 such as subtilisin BPN′ and subtilisin 309. Therefore, the reports do not provide useful information for a group of serine proteases having a gap region and an extended region of the C-terminus, which do not exist in the group of proteases having a molecular weight of approximately 28,000, and having a molecular weight of approximately 43,000.
Moreover, it is impossible to predict whether an alteration of amino acid residues in order to improve detergency of an enzyme will anyhow affect protein productivity or not. Therefore, even though the introduction of a certain mutation can bring a favorable change on detergency, the actual production may be extremely inhibited if it lowers its protein productivity.    [Patent Document 1] WO99/18218    [Patent Document 2] Japanese Patent No. 3343115    [Non-patent Document 1] Saeki et al., Biochem. Biophys. Res. Commun., Vol. 279, pp. 313-319, 2000    [Non-patent Document 2] Nature, Vol. 318, pp. 375-376, 1985    [Non-patent Document 3] J. Mol. Biol., Vol. 193, pp. 803-813, 1987    [Non-patent Document 4] Nature, Vol. 328, pp. 496-500, 1987    [Non-patent Document 5] Protein Engineering, Vol. 10, pp. 627-634, 1997